In the past few years, a considerable number of case reports describing predominantly gastrointestinal, urinogenital, and ophthalmic manifestations of microsporidial infections in acquired immune deficiency syndrome patients have been published. Although anecdotal evidence of the therapeutic efficacy of a number of antimicrobial agents has appeared in the literature, there has been little or no in vitro data to substantiate these claims. Of the principal species of microsporidia that are human pathogens, all but Enterocytozoon bienusi (E. bienusi) have been propagated in cell culture.We continue to look for ways to enhance the propagation of microsporidia in vitro. The current cell culture systems are inefficient. We have evaluated a number of cell lines and culture conditions to optimize growth of microsporidia in our assay. Efforts will continue to find a cell culture system for the propagation of E. bienusi. Once these objectives are achieved, we intend to screen potentially useful antimicrobial agents to determine which, if any, have deleterious effects on microsporidial replication, and if this inhibition is species specific. Compounds will also be examined for their ability to prevent infection of cell culture monolayers by microsporidia.Various laboratory methods will be used to evaluate the effectiveness of antimicrosporidial agents. Quantitation of released spores from established cultures will provide information concerning the effect of agents on microsporidial replication and spore production. A monoclonal antibody produced by Dr. Nash (NIAID, LPD) will be used to stain and count infected cells to determine inhibition of infection by antimicrobial agents. We have developed a reliable assay for quantitation that uses polymerase chain reaction and a europium-labeled DNA probe. Quantitation of microsporidial DNA will provide information about the ability of agents to prevent microsporidial replication within the host cell and the ability of agents to prevent infection of cell host cells.